Lysis of the host cell by bacteriophage is, as the most frequent cytocidal event in the biosphere, a truly fundamental process. In addition, understanding the molecular basis of phage lysis is now clinically relevant because phage therapy is emerging as an important tool against multi-drug resistant bacterial infections. There are two general modes: Multi-Gene Lysis (MGL), used by dsDNA phages, and Single-Gene Lysis (SGL), used by small single-strand nucleic acid phages. At minimum, MGL systems require a muralytic enzyme, the endolysin, that degrades the cell wall or peptidoglycan (PG), and a small membrane protein, the holin, that actively programs the function of the endolysin. At least 10 more classes of phage lysis proteins have also been identified, including spanins functioning in destruction of the outer membrane in Gram-negative infections or acting as regulators of holin and endolysin function. The lysis pathways have steps that both respond to and cause biophysical changes in the host membrane, as well as featuring multiple examples of dynamic membrane topology and massive quaternary rearrangements, ultimately resulting in holes in the bacterial membrane of unprecedented micron-scale. Overall, these complex MGL systems make lysis a precisely-controlled, all-or- nothing phenomenon. In contrast, the small ssDNA and ssRNA phages have no genomic room for MGL systems. Instead a single Sgl (single gene lysis) protein acts to cause dysfunction in host PG biosynthesis or homeostasis, eventually leading to a host autolysis. One class of Sgl?s that block steps in cell wall biosynthesis has been established and designated as Protein Antibiotics, but the target of more than 20 other Sgl?s identified by bioinformatics and phage genetics is not known. In the next five years, the focus will not only be on the remarkable spanins, which fuse membranes during lysis, but also on two new classes of MGL proteins: releasins and disruptins. Releasins are unique in licensing dynamic membrane topology of endolysins. Disruptins are small, amphipathic proteins that are used to weaken the outer membrane; surprisingly, when purified and used in vitro, they function as phage-encoded versions of the cationic antimicrobial peptides (CAMPs) produced by mammalian cells. The unique power of phage genetics will be used to determine the mechanisms of both these new MGL proteins. Our biophysical and structural collaborators will be supp;oed with mutants, phenotypes and constructs to be used in characterizing lysis at both the atomic level and in the context of the infected single cell. In the SGL area, the recent hyper-expansion of the metagenomics of ssRNA phages will be exploited to solve the targets of many new Sgl proteins. The hypothesis is that ssRNA phage Sgl proteins have evolved to attack every step in host cell wall synthesis and homeostasis. Also, a new model that a major class of Sgl?s acts by binding the universal cell wall precursor, Lipid II, will be tested.